PROJECT SUMMARY Heart failure (HF) remains a serious public health concern despite recent advances in medicine. Currently available drugs only address symptoms of HF, thus new approaches for curative therapies are sorely needed. Gene therapy has been proposed as one such promising approach; unfortunately, many candidate genes would lead to serious clinical side effects if delivered systemically. Additionally, considerable loss of delivery vectors to off-target organs require high vector doses to be used in order to achieve therapeutic effect at diseased tissue sites. To address these challenges, we propose to engineer adeno-associated virus (AAV) vectors that can specifically target cardiac tissue damaged after a myocardial infarction (MI). The key scientific premise of this project is the observation that extracellular proteases, specifically matrix metalloproteinases (MMPs) are elevated in damaged cardiac tissue post-MI. We have developed a platform of protease- activatable AAV vectors that can deliver genes in response to the MMPs elevated post-MI. Promisingly, upon intravenous injection, our engineered AAV vectors are able to achieve significantly improved targeted gene delivery to the high MMP region of the diseased heart in vivo, and this targeted delivery is accompanied by decreases in delivery to non-target organs. In this R01 project, we aim to design, build, and characterize an improved panel of protease-activatable AAV vectors for HF treatment. In aim 1, we will create AAV vectors that can target different disease stages post-MI. In aim 2, we will use molecular modeling and structural approaches to study the AAV capsid variants and to further improve our vector designs. Then in aim 3, we will use in vivo molecular imaging to characterize the in vivo specificity of the engineered vectors in relation to elevated MMP levels in the heart post-MI. Finally, in aim 4 we will test the therapeutic efficacy of using the protease-activatable AAV vectors in in vivo models of MI-induced HF. Overall, by improving the specificity of AAV vectors for target cardiac tissues, we aim to (i) overcome the need to use invasive administration strategies; (ii) minimize delivery to off-target organs, leading to decreased side effects as well as decreased overall vector dosage needed to achieve therapeutic effect, and (iii) reduce any dose-dependent immune responses against the vector.